The Anopheles gambiae complex contains the most efficient malaria vectors in the world. Identification of the species and the concomitant distribution are vital for effective malaria control. The objective of the study was to establish the diversity, spatial and seasonal abundance of malaria vectors in the Rufiji River Basin in Tanzania and the implications on malaria vector control. Mosquitoes were collected using CDC light-traps. Anopheles mosquitoes were identified by conventional keys. Anopheles gambiae complex were speciated using standard PCR method. Of the 562 specimens analysed by PCR 69% produced fragments equivalent to An. gambiae s.s. (390 bp), 23% equivalent to An. arabiensis (315bp), and 7% as An. merus (464 bp). An. gambiae s.s. and An. merus were more abundant on the plateau than on the flood plain (Fisher’s exact test, P< 0001), whereas An. arabiensis was equally abundant between the two sites (Fisher’s exact test, P=1656). The density of the three sibling species of An. gambiae complex also varied with the seasons. An. gambiae s.s. and An. arabiensis were most predominant species during the start of the rainy season, but as season progresses, An. gambiae s.s. predominated. An. merus was only recorded during the short rainy season. In conclusion, both An. gambiae s.s., An. arabiensis and An.merus are malaria vectors in the Rufiji River basin and that An. merus is recorded for the first time in the south-eastern coast of Tanzania. These findings are important in the planning and implementation of malaria vector control activities in the Rufiji River basin, south-eastern Tanzania.